The leading cause of pulmonary and cerebral embolism is thrombophlebitis. Thromboembolism is caused by abnormalities of the blood vessel, the blood or the circulation resulting the formation of blood clots called thrombi. Within a short period of time, many of these blood clots become dislodged from the blood vessels and migrate freely as emboli via the circulation into the lung. These emboli are then trapped and cause serious complications interfering with normal circulation and gas exchange.
Formation of blood clots occurs frequently in myocardial infarction and neoplasm. Recent experimental findings have confirmed the existence of fibrin or clot deposits at the sites of infarcts and tumors. Due to microvascular damage or injury, the coagulation process is initiated in the early or acute phase of these diseases resulting in a well formed fibrin gel surrounding the injured tissue or tumors. The fibrin clot effectively cuts off blood supply to the area of infarction and further aggravates the patient's symptoms. In case of neoplasm, the invading tumor utilizes the fibrin gel as a protective shield against antibody activity from the host thus maintaining its rapid growth without hinderence.
Early diagnosis of thromboembolic diseases remains a considerable problem. Clinical diagnosis of venous thrombosis are neither specific nor reliable during the acute phase of thrombophlebitis and phlebography is an invasive, uncomfortable technique. Human fibrinogen labeled with the radionuclide .sup.125 I has been used for screening high risk or postoperative patients. Because of low energy gamma photon flux and necessarily limited doses of .sup.125 I, the use of radioiodinated fibrinogen is limited to surface monitoring technics. It is not a scintillation imaging agent. The screening procedure requires 7-10 days. Autologous human fibrinogen labeled with .sup.123 I or .sup.131 I has been advocated recently as thrombus imaging agents. These agents are still in early experimental stages and are not available for general medical use.
Attempts to localize and to detect thrombus using Technetium-99m labeled streptokinase, urokinase(Dugan, MA U.S. Pat. No. 3,812,245) and fibrinogen(Abramovici, et at, U.S. Pat. No. 4,057,617) have had little success. Since the optimal condition for preserving the physiobiological properties of proteins occurs within a narrow pH range of 7 to 7.4, proteins that are labeled with .sup.99m Tc by the acidic method of Dugan(pH less than 2) or alkaline method of Abramovici(pH 11.6) are completely denatured. This renders protein substances labeled by their methods unsuitable for biological and medical applications. The existence of protein degradation products as described in their patents is further proof that proteins labeled at extremes of pH undergo drastic molecular alteration or transformation. Thus, these so called Tc-99m labeled proteins as claimed by Dugan and Abramovici do not have the same properties as they had prior to the labeling process. The use of denatured heterologous proteins in man carries risk of antigenic reaction and hepatitis transmission.
Protein denaturation and complete loss of biological activity are the primary concerns in the labeling of human plasma proteins with Tc-99m. To resolve these problems, the present inventor has developed a simple chemical method of labeling these protein substances with Tc-99m under physiological condition(Wong, DW, Patent pending, Ser. No. 939,820 filed Sept. 5, 1978 and now U.S. Pat. No. 4,293,537, and Wong, DW, et al, Int. J. Appl. Rad. and Isotopes 29:251, 1978). The basic labeling methodology involves the production of a .sup.99m Tc(Sn)citrate complex species with high protein binding capacity at pH 7.4 prior to the addition of the protein. The actual labeling of the protein ligand with Tc-99m occurs at physiological conditions of pH, thus avoiding harsh treatment of the protein and preserving its biological properties. Experimental results have confirmed that plasma proteins such as figrinogen, antibodies or immunoglobulins, urokinase, thrombin and streptokinase-activated human plasmin labeled with Tc-99m by the present chemical process retain their natural physiological and immunological properties(Wong, DW, et al, J. Nucl. Med. 20:967, 1979).
Technetium-99m labeled plasma proteins which retain their natural biological properties after labeling are ideal scintillation imaging agents. Essentially, these radiolabeled protein substances will actively participate in the physiobiochemical processes in vivo in man or animals. Tc-99m autologous antibodies, for example, are immunologically active against specific antigens thus providing a simple, unique and highly specific means of detecting infectious lesions or tumors. Similarly, diseases such as venous and arterial thrombosis, pulmonary or cerebral embolism, myocardial infarction and tumors can be diagnosed using Tc-99m labeled antologous human fibrinogen, urokinase or streptokinase-activated human plasmin.
Technetium-99m labeled urokinase and streptokinase-activated human plasmin (plasmin-SK) offer additional advantages over Tc-99m or I-131 labeled fibrinogen. Among these are: (a) both fibrinolytic enzymes are safe and non-antigenic in man; (b) these enzymes are available commercially in pure form freed from possible hepatitis transmission; (c) they have been used clinically in patients for the treatment of thromboembolic diseases without serious side effects and (d) they are effective in localizing and detecting preformed or aged blood clots even at a latter stage of the disease.